Agrobacterium is a genus of Gram-negative bacteria that uses horizontal gene transfer to cause tumors in plants. Agrobacterium tumefaciens is the most commonly studied species in this genus. Agrobacterium is well known for its ability to transfer DNA between itself and plants, and for this reason it has become an important tool for plant improvement by genetic engineering.
Agroinfiltration is a method in plant biology to induce transient expression of genes in a plant or to produce a desired protein. In the method a suspension of Agrobacterium is injected into a plant leaf, where it transfers the desired gene to plant cells. The benefit of agroinfiltration when compared to traditional plant transformation is speed and convenience
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The Agrobacterium genus is quite heterogeneous. Recent taxonomic studies have reclassified all of the Agrobacterium species into new genera, such as Ruegeria, Pseudorhodobacter and Stappia, but most species have been reclassified as Rhizobium species.
Plant pathogen: A. tumefaciens causes crown-gall disease in plants. The disease is characterised by a tumour-like growth or gall on the infected plant, often at the junction between the root and the shoot. Tumors are incited by the conjugative transfer of a DNA segment (T-DNA) from the bacterial tumour-inducing (Ti) plasmid. The closely related species, A. rhizogenes, induces root tumors, and carries the distinct Ri (root-inducing) plasmid. Although the taxonomy of Agrobacterium is currently under revision it can be generalised that 3 biovars exist within the genus, A. tumefaciens or biovar 1, A. rhizogenes or biovar 2, and A. vitis or biovar 3. Strains within biovars 1 and 2 are known to be able to harbour either a Ti or Ri-plasmid, whilst strains of biovar 3, generally restricted to grapevines, can harbour a Ti-plasmid. Non-Agrobacterium strains have been isolated from environmental samples which harbour a Ri-plasmid whilst laboratory studies have shown that non-Agrobacterium strains can also harbour a Ti-plasmid. Many environmental strains of Agrobacterium do not possess either a Ti or Ri-plasmid. These strains are avirulent.
The plasmid T-DNA is integrated semi-randomly into the genome of the host cell (Francis and Spiker, 2005. Plant Journal. 41(3): 464.), and the virulence (vir) genes on the T-DNA are expressed, causing the formation of a gall. The T-DNA carries genes for the biosynthetic enzymes for the production of unusual amino acids, typically octopine or nopaline. It also carries genes for the biosynthesis of the plant hormones, auxin and cytokinins. By altering the hormone balance in the plant cell, the division of those cells cannot be controlled by the plant, and tumors form. The ratio of auxin to cytokinin produced by the tumor genes determines the morphology of the tumor (root-like, disorganized or shoot-like).
Agrobacterium in humans: Although generally seen as an infection in plants, Agrobacterium can be responsible for opportunistic infections in humans with weakened immune systems, but has not been shown to be a primary pathogen in otherwise healthy individuals. A 2000 study published by the National Academy of Sciences suggested that Agrobacterium attaches to and genetically transforms several types of human cells by integrating its T-DNA into the human cell genome. The study was conducted under laboratory conditions and states that it does not draw any conclusions regarding related biological activity in nature.
The ability of Agrobacterium to transfer genes to plants and fungi is used in biotechnology, in particular, genetic engineering for plant improvement. A modified Ti or Ri plasmid can be used. The plasmid is 'disarmed' by deletion of the tumor inducing genes; the only essential parts of the T-DNA are its two small (25 base pair) border repeats, at least one of which is needed for plant transformation. Marc Van Montagu and Jozef Schell at the University of Ghent (Belgium) discovered the gene transfer mechanism between Agrobacterium and plants, which resulted in the development of methods to alter Agrobacterium into an efficient delivery system for gene engineering in plants. A team of researchers led by Dr Mary-Dell Chilton were the first to demonstrate that the virulence genes could be removed without adversely affecting the ability of Agrobacterium to insert its own DNA into the plant genome (1983).
The genes to be introduced into the plant are cloned into a plant transformation vector that contains the T-DNA region of the disarmed plasmid, together with a selectable marker (such as antibiotic resistance) to enable selection for plants that have been successfully transformed. Plants are grown on media containing antibiotic following transformation, and those that do not have the T-DNA integrated into their genome will die. An alternative method is agroinfiltration.
Transformation with Agrobacterium can be achieved in two ways. Protoplasts, or leaf-discs can be incubated with the Agrobacterium and whole plants regenerated using plant tissue culture. A common transformation protocol for Arabidopsis is the floral-dip method: the flowers are dipped in an Agrobacterium culture, and the bacterium transforms the germline cells that make the female gametes. The seeds can then be screened for antibiotic resistance (or another marker of interest), and plants that have not integrated the plasmid DNA will die.
Agrobacterium does not infect all plant species, but there are several other effective techniques for plant transformation including the gene gun.
Genomics: The sequencing of the genomes of several species of Agrobacterium has permitted the study of the evolutionary history of these organisms and has provided information on the genes and systems involved in pathogenesis, biological control and symbiosis. One important finding is the possibility that chromosomes are evolving from plasmids in many of these bacteria. Another discovery is that the diverse chromosomal structures in this group appear to be capable of supporting both symbiotic and pathogenic lifestyles. The availability of the genome sequences of Agrobacterium species will continue to increase, resulting in substantial insights into the function and evolutionary history of this group of plant-associated microbes.
Agrobacterium tumefaciens is the causal agent of crown gall disease (the formation of tumours) in over 140 species of dicot. It is a rod shaped, Gram negative soil bacterium (Smith et al., 1907). Symptoms are caused by the insertion of a small segment of DNA (known as the T-DNA, for 'transfer DNA') into the plant cell, which is incorporated at a semi-random location into the plant genome.
Agrobacterium rhizogenes is a Gram negative soil bacterium. In the rhizosphere, plants may suffer from wounds by soil pathogens or other sources. This leads to the secretion of phenolic compounds like acetosyringone which have chemotactic effects that attract the bacteria. Under such conditions, certain bacterial genes are turned on leading to the transfer of its T-DNA from its Ri plasmid into the plant through the wound. After integration and expression, in vitro or under natural conditions, the hairy root phenotype is observed.
Ti plasmid is a circular plasmid that often, but not always, is a part of the genetic equipment that Agrobacterium tumefaciens and Agrobacterium rhizogenes use to transduce its genetic material to plants. The Ti plasmid is lost when Agrobacterium is grown above 28C. Such cured bacteria do not induce crown galls, i.e. they become avirulent. pTi and pRi share little sequence homology but are functionally rather similar. The Ti plasmids are classified into different types based on the type of opine produced by their genes. The different opines specified by pTi are octopine, nopaline, succinamopine and leucinopine.
Agroinfiltration is a method in plant biology to induce transient expression of genes in a plant or to produce a desired protein. In the method a suspension of Agrobacterium is injected into a plant leaf, where it transfers the desired gene to plant cells. The benefit of agroinfiltration when compared to traditional plant transformation is speed and convenience.
First step of the protocol is to introduce a gene of interest to a strain of Agrobacterium. Subsequently the strain is grown in a liquid culture and the resulting bacteria are washed and suspended into a buffer solution. This solution is then placed in a syringe (without a needle). The tip of the syringe is pressed against the underside of a leaf while simultaneously applying gentle counterpressure to the other side of the leaf. The Agrobacterium solution is then injected into the airspaces inside the leaf through stomata, or sometimes through a tiny incision made to the underside of the leaf.
Vacuum infiltration in another way to penetrate Agrobacterium deep into plant tissue. In this procedure, leaf disks, leaves, or whole plants are submerged in a beaker containing the solution, and the beaker is placed in a vacuum chamber. The vacuum is then applied, forcing air out of the stomata. When the vacuum is released, the pressure difference forces solution through the stomata and into the mesophyll.
Once inside the leaf the Agrobacterium transforms the gene of interest to a portion of the plant cells. The gene is then transiently expressed. The plant can be monitored for a possible effect in the phenotype, subjected to experimental conditions or harvested and used for purification of protein of interest. Many plants can be transformed by this method, but the most common ones are Nicotiana benthamiana and Nicotiana tabacum.
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